HCN1-WT (15)	−57.3 ± 1.3*	−63.2 ± 1.0	307 ± 35	. . .	. . .	. . .
HCN1-Ins (16)	−59.9 ± 1.0*	−65.9 ± 0.9	321 ± 29	. . .	. . .	. . .
HCN1-ΔΔΔ (12)	−61.3 ± 1.2*	−68.3 ± 0.8	266 ± 35	. . .	. . .	. . .
HCN1-ΔΔΔ_ZD7288 (8)	−72.0 ± 1.2	. . .	286 ± 38	. . .	. . .	. . .

Spontaneously firing cells

HCN1-ΔΔΔ (19)	. . .	−60.6 ± 0.5†	239 ± 23	206 ± 16†	306 ± 32†	85.1 ± 8.6†
HCN1-ΔΔΔ + ZD7288	−69.3 ± 0.4	. . .	261 ± 37	. . .	. . .	. . .
(8)
Control + BaCl2 (10)	. . .	−46.3 ± 2.6	296 ± 34	92 ± 15	46.2 ± 7.6	465 ± 118

All values are mean ± SEM. Numbers in parenthesies represent the numbers of determinations.
*Statistically different (p < 0.01) from control.
†Statistically different (p < 0.01) from Ba²⁺cells.

Next, the functional consequences of Ad-CGI-HCN1—, Ad-CGI-HCN1-Ins-, and Ad-CGI-HCN1-ΔΔΔ-mediated I_foverexpression in the AP waveform of LVCMs was investigated (FIG. 9). Interestingly, automaticity was exclusively observed in 61% of Ad-CGI-HCN1-ΔΔΔ-transduced cells recorded (n=19 of 31), even without I_K1inhibition and pulse stimulation, but never in control LVCMs. The AP firing rate of electrically active Ad-CGI-HCN1-ΔΔΔ-transduced LVCMs was 206±16 bpm, comparable to the native guinea pig heart rate and much higher than that induced by Ba²⁺ (P<0.01). The AP duration to 50% repolarization (APD₅₀) (239±23 ms) was not different from control LVCMs (Table 1; P>0.05). Of note, the MDP (−60.6±0.5 mV; n=19) was significantly depolarized relative to the RMP of control cells (−70.6±0.6 mV; n=8; P<0.01) and associated with agradual phase 4 depolarization (slope=306±32 mV/s). These properties were typical of genuine nodal cells. Nonetheless, the rapid AP upstroke (V=86±8 V/s; n=13) and overshoot observed were indicative of the ventricular origin of these rhythmic “pacemaker-like” cells. These in vitro data were in complete accordance with the recently reported in vivo experiments (71).

To obtain mechanistic insights, the effect of ZD7288 on Ad-CGI-HCN1-ΔΔΔ-transduced cells was studied. Interestingly, both the induced I_fand AP firing could be blocked by ZD7288 (FIG. 9A). Indeed, WT RMP also was restored (−69.3±0.4 mV; n=8; P>0.05) (FIG. 9B). Interestingly, when given a depolarizing stimulus, silenced Ad-CGI-HCN1-ΔΔΔ-transduced cells could once again elicit normal single APs (FIG. 9B). All AP parameters before and after I_fblockade are summarized in Table 1. Collectively, these data indicated that Ad-CGI-HCN1-ΔΔΔ-mediated AP firing was a direct result of the induced I_f. The following experiments were designed to mechanistically dissect specific I_fproperties that led to automaticity.

Intermediate Phase 4-Like Depolarization

In stark contrast to the pacemaker-like phenotype, the remaining 39% of Ad-HCN1-ΔΔΔ-transduced cells exhibited a depolarized RMP (−61.3±1.2 mV; n=12; P<0.01) but without spontaneous APs (as a result of a lower level of expressed I_fbecause of biological variation; see later); however, a single ventricular AP associated with an incomplete “phase 4-like” depolarization that did not lead to subsequent firing could be elicited on simulation (FIG. 9C). Addition of ZD7288 restored RMP to the normal hyperpolarized level (−72.0±1.2 mV; n=8) (FIG. 9C; Table 1). Furthermore, a normal ventricular AP also could be elicited on stimulation after I_fblockage by ZD7288 (FIG. 9C). Of note, the incomplete phase 4-like depolarization also disappeared. Such an intermediate phase 4-like phenotype, which could be reverted to control ventricular AP phenotype after ZD7288 blockade, was similarly observed in 100% of Ad-CGI-HCN- (FIG. 9D) and 100% of Ad-CGIHCN1-Ins-transduced LVCMs (FIG. 9E), indicating that overexpression of WT or Ins HCN1 channels clearly influences the RMP andphase 4 depolarization but was insufficient to lead to AP firing, unlike HCN1-ΔΔΔ.

To explore the basis of phase 4-like depolarization, the difference between RMPs before and after ZD7288 (ΔV_phase4) was plotted against the I_fmagnitude at −60 mV (the earliest voltage at which Ad-CGI-HCN1— and Ad-CGI-HCN1-Ins-induced I_fcould first be recorded) of Ad-CGI-HCN1-, Ad-CGI-HCN1-ΔΔΔ-, and Ad-CGI-HCN1-Ins-transduced LVCMs (FIG. 9F). The data were well fitted with a single exponential function, although statistically different plateau values were reached for the 3 groups (20.9±1.2, 15.5±1.8, and 12.0±1.5 mV, respectively; P<0.01). Of note, the rank order parallels the corresponding activation midpoints.

Adrenergic Stimulation of Spontaneously Firing Ad-CGI-HCN1-ΔΔΔ-Transduced LVCMs

To further characterize spontaneously-firing Ad-CGI-HCN1-ΔΔΔ-transduced LVCMs as a pacemaker, their response to adrenergic stimulation by superfusion of 1 μmol/L isoproterenol for 5 minutes was studied. The cycle length reduced from 234±5.5 to 182±4.1 ms (P<0.01; n=4), representing a 27% increase in firing rate (FIGS. 10A and 10B). This acceleration could be attributed largely to the change inphase 4 slope from 295±5 to 350±12 mV/s, which subsequently shortened APD (P<0.05;FIG. 10C); MDP also became more depolarized (from −60.3±0.4 to −55.5±0.7 mV; P<0.05;FIG. 10D). However, there was no significant change in the TOP after isoproterenol application (−45.2±0.4 versus −44.6±0.5 mV of control; P>0.05;FIG. 10E). On inspection of the steady-state activation curves recorded from the same cells, the midpoint (V_1/2) was positively shifted from −62.5±1.0 to −56.9±1.6 mV (P<0.05;FIG. 10F).

Effects of Acetylcholine on Spontaneous AP Firing

Furthermore, the effect of acetylcholine on the pacing parameters of spontaneously firing Ad-CGI-HCN1-ΔΔΔ-transduced LVCMs was studied. On application of acetylcholine (1 μmol/L) to the bath solution, there were no significant changes in cycle length (293±17 versus 293±16 ms; n=6; P>0.05), MDP (−59.2±0.9 versus −59.0±1.0 mV; P>0.05), andphase 4 slope (236±15 to 241±16 mV/s) from baseline to acetylcholine (20 seconds;FIG. 10G).

Effects of Tetrodotoxin on Spontaneous AP Firing

On application of the Na channel-specific blocker tetrodotoxin (TTX; 60 μmol/L) to electrically active Ad-CGI-HCN1-ΔΔΔ-transduced LVCMs (n=4), there was a notable increase in cycle length from 256±11 to 328±23 ms (FIGS. 11A and 11B). This increase was associated with a decrease in the maximal upstroke velocity of AP from 68.8±8.3 to 5.3±0.6 V/s (P<0.01), as well as a depolarized TOP from 43.7±0.4 to 27.0±3.4 mV (P<0.05;FIGS. 11C and 11D). Of note, the AP overshoot also disappeared. Despite these changes, there was no statistical difference in thephase 4 slope and MDP (FIGS. 11E and 11F), consistent with the notion that I_fwas unaltered in these experiments.

Effects of Overdrive Pacing

To test the response to overdrive pacing, a train of pulse stimulation of 0.1 to 1 nA for 5 ms at 6.7 Hz (ie, cycle length, 150 ms or 400 per minute) was applied to electrically active Ad-CGI-HCN1-ΔΔΔ-transduced LVCMs for 30 seconds (n=6) in a manner similar to that previously used to study overdrive suppression in rabbit SA nodal cell (89). A representative experiment is shown inFIG. 12A.FIG. 12B shows on an expanded scale overlapping APs recorded before, during and after overdrive stimulation. The cycle length significantly shortened during the stimulation, indicating that the ability of Ad-CGI-HCN1-ΔΔΔ-induced biopacemaker to adapt to the rate remained intact. However, there were no detectable changes inphase 4 slope, MDP, and TOP (FIG. 12C; P>0.05). These parameters are summarized in Table 2.

TABLE 2


Effects of Overdirve Pacing on AD-CGI-HCN1-ΔΔΔ Spontaneously
Firing Cells

	Baseline	After Overdrive	P

Pacing rate, bpm	217 ± 13	214 ± 15	0.32
MDP, mV	−61.3 ± 0.4	−61.6 ± 0.7	0.94
Phase 4 slope,	157 ± 12	157 ± 13	0.99
mV/s
TOP, mV	−48.1 ± 2.4	−43.7 ± 3.4	0.07

All values are means ± SEM (n = 6 from a single animal).

What Specific I_fParameters Underlie the Induced Automaticity?

To mechanistically dissect the role of I_fin spontaneous AP firing, correlation analysis was performed to examine in details the relationships between various AP parameters and I_fproperties.FIG. 13 shows that for electrically active Ad-CGI-HCN1-ΔΔΔ-transduced LVCMs, the induced pacing rate (A), MDP (B), andphase 4 slope (C) were all positively correlated to the I_fdensity at −50 mV (r=0.94, 0.70, and 0.81, respectively). Whereas net outward whole-cell currents of LVCMs (from 40 to 70 mV) tend to hyperpolarize the cells, Ad-CGI-HCN1-ΔΔΔ-transduced cells had zero or inward currents over the same voltage range (FIG. 14A), which tend to depolarize the cells. All these in turn translated into a significantly smaller rectification ratio (FIG. 14B).

When the net total currents at 50 mV ([I_f+I_K1]_{−50 mV}) of control and Ad-CGI-HCN1-ΔΔΔ-transduced cells were plotted against the corresponding RMP or MDP (FIG. 14C), an interesting pattern emerged: RMP or MDP became more depolarized with increasingly less positive (I_f+I_K1)_{−50 mV}, consistent with that presented inFIG. 13B; furthermore, net negative (I_f+I_K1)_{−50 mV}occurred only in spontaneously firing cells (ie, 61% of Ad-CGI-HCN1-ΔΔΔ-transduced cells; squares inFIG. 14), never in the quiescent ones (including control cells, 39% of Ad-CGI-HCN1-ΔΔΔ-transduced cells, and electrically active Ad-CGI-HCN1-ΔΔΔ-transduced LVCMs that had been silenced by ZD7288, as represented by inverted triangles, circles, and triangles, respectively).

Discussion

Although I_fis most abundant in the SA node, the region that normally paces the entire heart, it is also found at various levels in cardiac tissues such as the atria and ventricles. Unlike rhythmic nodal pacemaker cells, however, adult atrial and ventricular cells are normally electrically quiescent unless they are stimulated by pacemaker activity arising elsewhere. This is due to the intense expression in the atria and ventricles of the cardiac inward-rectifier K⁺ current or I_K1, encoded by the Kir2 gene family, that stabilizes a negative resting membrane potential (≈80 mV) and thus suppresses any latent spontaneous electrical activity while maintaining the cells fully excitable. In contrast, I_K1, an “opponent” of I_f, is absent in nodal pacemaker cells, rendering the latter more prone to oscillations. Miake and colleagues (36, 37) have demonstrated that latent pacemaker activity of normally silent ventricular myocytes can be unleashed to produce spontaneous firing activity by genetic inhibition of Kir2-encoded I_K1. However, I_K1suppression-induced pacemaking activity is ≈3 times slower than normal, indicating that other genuine, active players are involved in pacemaking nodal cells. The present study is focused on dissecting the mechanistic role of I_f.

The experimental results presented in this study fully support the notion that I_fis a crucial, intrinsic oscillator, rather than just a secondary modulator, of the membrane potential. However, its contribution is heavily dependent on the relative expression level of I_K1. Mechanistically, the RMP is ≈−71 mV when I_K1dominates and the net current is outward with an amplitude of at least 1.6 pA/pF (ie, [I_f+I_K1]_{−50 mV}>1.6 pA/pF). However, when the net outward current is <1.6 pA/pF (ie, 0 pA<[I_f+I_K1]_{−50 mV}<1.6 pA/pF) because of increasing levels of I_fexpression, I_fpartially counterbalances I_K1and thereby depolarizes the RMP (up to ≈62 mV) or even drive membrane depolarization, leading to the incomplete, phase 4-like slope observed in the intermediate AP phenotypes (cfFIG. 9C through 9E). Within the range of 0 to 1.6 pA/pF, I_famplitude is proportional to the rising speed of phase 4-like depolarization (FIG. 13C) but is still insufficient to cause electrical oscillations. When I_fis sufficiently expressed to dominate I_K1so that the net current becomes inward (ie, [I_f+I_K1]_{−50 mV}<0 pA/pF), spontaneous firing can be induced. Once induced, both the firing rate (FIG. 13A) and MDP (FIG. 13B) are directly proportional to I_f(up to at least 6 pA/pF).

In recent years, various gene- and cell-based approaches have been tested to generate a functional biological pacemaker (74-76, 78, 79, 88-93) as a potential replacement for or supplement to conventional electronic pacemakers that are commonly used to treat impulse generation defects (eg, bradycardias). Understanding the mechanistic action of I_fin cardiac automaticity is invaluable for achieving this goal. Based on the substantial groundwork previously done by others, the recent in silico analysis, (48) in vivo experiments, (71) and present mechanistic study clearly indicate that genetic induction of pacing and, more important, its fine-tuning, can be achieved by tuning the relative activity of I_fand I_K1. This can perhaps be accomplished best by custom tailoring the activation gating properties of I_fvia engineering of such HCN channel functional motifs as the S3-S4 linker (75) or the S4 segment.

Of note, although I_fand I_K1counterbalance each other within a certain voltage range, I_K1also hyperpolarizes the RMP, which in turn can result in a higher probability of opening for the so-called hyperpolarization-activated I_fchannels. This is particularly true under the dynamic conditions of pacing as a result of hysteretic properties of HCN channels (48). Therefore, the strategies of I_K1inhibition and I_foverexpression to induce pacing in normodal cardiomyocytes, which carry distinct compositions of other ion channel types, are not necessarily synergetic. Clearly, a fine balance between the 2 is needed for the most desirable outcome.

Because biological pacemakers are powered by passive transports driven by the ionic gradients, they do not require battery replacement like electronic pacemakers. More important, another major advantage over the electronic counterpart is the ability to maintain the in vivo responsiveness of pacing to endogenous neuronal and hormonal inputs. Although individual isoforms exhibit different sensitivities to cAMP modulation (mediated via the cyclic nucleotide-binding domain (90)), the response of I_f-induced biological pacemaker to sympathetic and parasympathetic agents can likewise be engineered. In fact, individual amino acid substitutions designed to alter gating and cAMP sensitivity can be combined simultaneously to achieve a particular phenotype. In other words, HCN-based biopacemaker can be “programmed,” unlike the binary nature of I_K1suppression.

The SA node is undoubtedly a complex tissue consisting of a heterogeneous population of pacemaker cells. For instance, there are gradual changes in such phenotypic properties as AP profile, (91, 92) ionic current densities, (62) and gap junction expression (62) from central (dominant or the leading pacemaker site) to peripheral (subsidiary) nodal cells. These differences and anatomic arrangements ensure that the leading center cells are protected from any overhyperpolarizing effects from the surrounding mass of atrial cardiomyocytes and that the depolarization wave front is propagated in the proper directions. An interesting feature of the Ad-CGI-HCN1-ΔΔΔ-induced biopacemaker is the lack of overdrive suppression, mimicking the native peripheral nodal cells (93). In the presence of TTX, AP firing exhibits such features of peripheral nodal cells as an increased cycle length, a depolarized TOP, and a slowed AP upstroke (94). Although it is well known that L-type Ca²⁺ current plays an obligatory role in pacemaking of both central and peripheral nodal cells, (94) Ca²⁺ and Na⁺ loads have been demonstrated to lead to overdrive excitation and suppression, respectively (95). The contribution of these mechanisms to the observed phenotypes requires further studies. Nevertheless, the present results imply that efforts may now focus on other ionic currents and electrogenic pumps to fine-tune I_f-induced pacemaking activity and other properties essential for the SA node to function properly.

EXAMPLE 3

I_f, or ‘funny’ current, encoded by the hyperpolarization-activated cyclic nucleotide-modulated (HCN) channel gene family, plays a pivotal role in cardiac pacing by acting as an intrinsic oscillator that drives diastolic depolarization to the action potential (AP) threshold after each excitation cycle (63, 64, 102). The expression of this current and the absence of a resting membrane potential (RMP) stabilizer, the inwardly rectifying potassium current I_K1) (62), are signatures of pacemaker cells that make up the sino-atrial (SA) node. Conversely, I_K1but not I_f, is robustly expressed in the silent-yet-excitable adult atrial (A) and ventricular (V) cardiomyocytes (CMs) (101, 103). Based on these biophysical characteristics, two genetic approaches, I_K1suppression (36, 59) and I_foverexpression (69, 96, 100, 102), have been independently experimented to convert normally quiescent CMs into spontaneously AP-firing cells as bio-artificial pacemakers (97). Although the two strategies appear to be conceptually synergistic, their complementarity has not been investigated. Furthermore, previous cellular and electrophysiological experiments were largely limited to VCMs rather than ACMs, even though the anatomical location of the SA node makes ACMs the more likely recipient for future gene-based therapies of heart rhythm disorders (e.g. sick sinus syndrome). Using a combination of computational modeling and somatic gene transfer techniques, the biophysical basis and interactions of I_fand I_K1in the induction of atrial automaticity was explored.

Materials and Methods

Mathematical Formulation

This mathematical model was formulated and modified based on a previous guinea pig ventricular model (99), with ion concentrations fixed for simplicity. I_fand I_K1were introduced and adjusted as previously described (48, 98). Numerical results were obtained from Matlab using a variable order ordinary differential equation solver plus a built-in backward-difference method, with a relative tolerance of 10⁻⁸and an absolute tolerance of 10⁻⁴.

Molecular Biology

PCR-based mutagenesis of mouse HCN1 (generously provided by Dr. Steve Segalbaum, Columbia University) of the bicistronic adenovirus shuttle vector pAdCMV-GFP-IRES (or pAdCGI) was performed with overlapping oligos as described in our previous publications (74, 75). The internal ribosomal entry site (IRES) allows the simultaneous translation of two transgenes, GFP and an engineered-HCN1 construct, with a single transcript. HCN1 was chosen because its biophysical properties were most extensively characterized in our previous reports (82, 80, 48, 81, 74, 75, 78, 79, 102), making it the best candidate for gene transfer experiments. Adenoviruses were generated by Cre-lox recombination of purified μ5 viral DNA and shuttle vector DNA using Cre4 cells (46). The recombinant products were plaque purified, amplified, and purified again by CsC1 gradients, yielding concentrations on the order of 10¹⁰plaque-forming units (PFU) ml⁻¹.

Adenovirus-Mediated Gene Transfer and Isolation of ACMs

Adult female guinea pigs (˜250 g) were euthanized by intra-peritoneal injection of pentobarbital (80 mg/kg). The hearts were quickly excised, followed by perfusion with 200 U/ml collagenase II using a customized Langendorff apparatus (84). ACMs were plated at 5×10⁵per laminin-coated glass coverslips in medium containing: 5 mM carnitine, 5 mM creatine, 5 mM taurine, 100 μg ml⁻¹penicillin-streptomycin and 10% fetal bovine serum in Medium 199 (Invitrogen Corp., CA, USA) at 37° C. with 5% CO₂for 2 hours. For transduction, ACMs were incubated for 1 hour in serum-free medium containing adenoviral particles at a concentration of ˜2×10⁹PFU ml⁻¹. A transduction efficiency of 70-80% could typically be achieved with this protocol.

Electrophysiology and Data Analysis

Electrophysiological experiments were performed using the whole-cell patch-clamp technique at 37° C. with an Axopatch 200B amplifier and pClamp 9.2 software (Axon Instruments Inc., CA, USA). A xenon arc-lamp was used to view GFP fluorescence at 488/530 nm (excitation/emission). Successfully transduced cells showed green epifluorescence. Patch pipettes were prepared from 1.5 mm thin-walled borosilicate glass tubes using a Sutter micropipette puller P-97 and had typical resistances of 3-5 MΩ when filled with an internal solution containing (mM): 110 K-aspartate, 20 KCl, 1 MgCl₂, 0.1 Na-GTP, 5 Mg-ATP, 5 Na₂-phosphocreatine, 1 EGTA, 10 HEPES, pH adjusted to 7.3 with KOH. The external Tyrode bath solution consisted of (mM): 140 NaCl, 5 KC1, 1 MgCl₂, 1 CaCl₂, 10 Glucose, 10 HEPES, pH adjusted to 7.4 with NaOH. Voltage- and current-clamp recordings were performed at ˜37° C. within 24 to 36 hours after culturing. To avoid contaminations of automaticity due to time-dependent I_K1reduction in culture (85), control ACMs were recorded during the same time window. Our control experiment showed that I_K1density at −100 mV was not significantly different between cultured and freshly isolated ACMs (−10.6±1.0pA/pF vs. −11.8±2.9 pA/pF, p>0.05).

To elicit inward currents, ACMs were held at −30 mV and pulsed from 0 to −140 mV with 10 mV increments for 2s, followed by a 1s, −100 mV pulse. I_fwas defined as 20 μM ZD7288-sensitive, 3 mM Ba²⁺-insensitive, time-dependent currents whereas I_K1was defined as a 3 mM Ba²⁺-sensitive current. For action potential recordings, ACMs were held at 0 pA without (for electrically active ACMs) or with a stimulation of 0.1-1 nA for 5 ms to elicit a response. Measurements of APs during pharmacological block of either I_for I_K1were performed with ACMs maintained at 0 pA without stimulation.

The steady state current-voltage (I-V) relationship was determined by plotting the currents measured at the end of a 2s test pulse of the protocol mentioned above against the test potentials. The voltage dependence of HCN channel activation was assessed by plotting time-dependent tail currents at −100 mV measured immediately after the 2s test pulse (0 to −140 mV) as a function of the test pulse voltage. Currents were normalized to the maximum tail current recorded. These recordings were made in the presence of 3 mM BaCl₂to block I_K1.

The data reported were mean ±SEM (uncorrected for the junction potential of −15.1 mV) with p<0.05 indicating statistical significance as determined by an unpaired t-test.

Results

I_fOverexpression Silences Instead of Inducing Automaticity

In the absence of I_fbut presence of I_K1, the baseline simulations reproduced the quiescent phenotype of adult VCMs with a RMP close to the reversal potential of K⁺ (FIG. 15A left). Upon receiving a current stimulus, a typical adult AP could be elicited (FIG. 15B). With gradual increase in the maximum conductance for I_f(from G_If=0 pS) while keeping constant conductance for I_K1spontaneous firing was achieved, consistent with what was mathematically (48) and experimentally (71, 102) demonstrated previously. Increase of conductance for I_f(from G_If=5 to 7.5 nS) subsequently accelerated the firing frequency, which was associated with a reduced AP amplitude, a depolarized maximal diastolic potential (MDP) and a diastolic phase 4-depolarizing phase that were similar to those of genuine adult nodal pacemaker cells (FIG. 15A middle). Interestingly, rather than accelerating the firing frequency, further increase in I_f(G_If>20 nS) significantly depolarized RMP and subsequently led to cessation of automaticity (FIG. 15A right). Unlike the normal ventricular phenotype, however, a single electrical stimulus gave rise to damped oscillations of the membrane potential (FIG. 15C).

I_fOverexpression in ACMs Resulted in Distinct Electrical Phenotypes

To explore in details the basis of atrial automaticity induction, I_fwas introduced into ACMs by transduction with the recombinant adenovirus Ad-CGI-HCN1-ΔΔΔ. The engineered HCN1-ΔΔΔ construct, whose S3-S4 linker has been shortened by deleting residues 235-237 to favor channel opening, was chosen to reproduce native I_fwithout the need to consider such poorly defined factors as accessory subunits and cellular context as was recently described (71, 74, 77). Control un-transduced ACMs had I_K1(FIG. 16a) but no I_f(FIG. 16B) while the transduced ACMs displayed robust I_K1(FIG. 16c) and I_f(FIG. 16B).

All control (un-transduced) ACMs were electrically quiescent but capable of generating a single AP upon the injection of a depolarizing current stimulus (0.1-1.0NA for 5 ms) (FIG. 17A). HCN1-ΔΔΔ transduction of ACMs resulted in two distinct electrophysiological phenotypes: an intrinsically firing and a quiescent-yet-excitable type. The first, accounting for 18% of all HCN1-ΔΔΔ-transduced ACMs recorded (n=113), exhibited rhythmic firing (FIG. 17B), even in the complete absence of any external stimulus. The spontaneous APs had a rate of 240±14 bpm, an upstroke velocity of 121±34 mV s⁻¹and a gradual phase 4-depolarization slope of 143±28 mV s⁻¹. Of note, their MDP averaged to −45.3±2.2 mV, and was significantly depolarized relative to the RMP of control ACMs at −58.5±1.0 mV (p.<0.01;FIG. 18A). The remaining 82% of the HCN1-ΔΔΔ-transduced ACMs were completely quiescent (FIG. 17C-D). Detailed analysis of these electrically silent ACMs post-transduction enabled further categorization into two subgroups: one with a mean hyperpolarized RMP of −56.7±1.3 mV (n=15 of 93) and another with a significantly more depolarized RMP around −27.6±1.3 mV (n=78 of 93;FIG. 18A). Electrical stimulation elicited from both groups a single AP displaying an incomplete “phase 4-like” depolarization that was unable to lead to subsequent firing (FIG. 17C-D). Such phase 4-like depolarization was more prominent in the group that had depolarized RMPs, showing a depolarization slope of 68±7 mV s⁻¹(vs. 27±5 mV s⁻¹of the group with a hyperpolarized RMP;FIG. 18B). In addition, their mean AP duration to 90% repolarization (APD₉₀) was 61.1±5.0 ms, longer than the hyperpolarized RMP group (APD₉₀=27.3±27.3 ms) although this difference did not reach statistical significance (p=0.31).

Successful In Vitro Conversion of HCN1-ΔΔΔ-Transduced ACMs Depends on the Relative Magnitude of I_fto I_K1

To explore the ionic basis underlying the different electrical phenotypes for Ad-CGI-HCN1-ΔΔΔ-transduced ACMs, I_fand I_K1was measured (FIG. 17E-H). Given the observed heterogeneity of AP profiles, voltage- and current-clamp recordings were simultaneously performed on the same cells to directly correlate I_fand I_K1magnitudes to the resultant AP phenotype. Quiescent control ACMs robustly expressed Ba²⁺-sensitive I_K1with the typical inwardly rectifying behavior but no I_f(FIG. 17E; n=12). Conversely, hyperpolarization-activated time-dependent I_fcould be seen in all HCN1-ΔΔΔ-transduced ACMs. The range of magnitudes varied due to the sporadic nature of Ad-mediated transgene expression (FIG. 17F-H). Despite these differences in I_f, there was no statistically significant I_K1density (at −140 mV) difference relative to control (−22.0±2.1 pA/pF) among spontaneously firing ACMs: (−23.3±6.5pA/pF, p=0.85), quiescent cells with depolarized RMP (−26.9±4.4pA/pF, p=0.34), and quiescent cells with hyperpolarized RMP (−30.6±3.8pA/pF, p=0.07). Of note, Ad-CGI-HCN1-ΔΔΔ-transduced ACMs that spontaneously fired APs had a sizable I_fbut its magnitude was only 50±17% of I_K1when assessed at −140 mY (FIG. 17F). Interestingly, transduced depolarized RMPs expressed comparable I_fand I_K1(FIG. 17G vs. 17H). I_fto I_K1ratios at −140 mV for all AP phenotypes are summarized inFIG. 18C.

Since the computational and experimental results strongly suggest that automaticity occurs only within a finite range of I_f/I_K1ration, experimental proof to the counter-intuitive notion that I_K1suppression may cease spontaneous firing, whereas I_fsuppression may induce automaticity was explored.FIG. 19A shows that in a typical spontaneously firing HCN-ΔΔΔ-transduced ACMs (n=4), I_K1suppression with 3 mM Ba²⁺ to increase the I_f/I_K1ratio paradoxically terminated the rhythmic firing and shifted the RMP to −0.8±0.2 mV. Indeed, application of Ba²⁺ to HCN-ΔΔΔ-transduced ACMs with hyperpolarized RMP, whose I_f/I_K1ratio at −140 mV was smaller than the spontaneously firing HCN-ΔΔΔ transduced ACMs (FIG. 18C), induced transient automaticity as their RMP depolarized and achieved an optimal I_f/I_K1(FIG. 19B; n=9). Interestingly, in quiescent HCN-ΔΔΔ-transduced ACMs with depolarized RMP (I_f/I_K1˜1 at −140 mV;FIG. 18C), I_fsuppression (to lower the ratio) with 20 μM ZD7288, a specific HCN channel blocker, shifted the RMP from −29.2±0.8 mV to −47.6±1.2 mV and subsequently triggered rhythmic firing (FIG. 19C; n=3). Collectively, this experiment shows that an optimal balance of I_fand I_K1, instead of a one-sided I_foverexpression or I_K1suppression, is necessary for automaticity induction and more importantly, its modulation.

Discussion

In recent years, efforts to create a bio-artificial pacemaker, an attractive alternative to electronic devices in treatment of cardiac arrhythmia, have experimented exclusively by either maximizing I_f(71, 96) or minimizing its “antagonizing” I_K1(36), to convert non-pacing CMs into spontaneously firing pacemaker cells. Through in silico and in vitro HCN-gene-transfer ACM experiments, the present study demonstrates that both I_fand I_K1play critical roles in automaticity induction and the new concept that a precise balance of these two currents, rather than a one-sided manipulation, is a prerequisite to induction.

Mechanistically, for any given current density of I_K1, introduced I_fcould induce automaticity only when its maximal current density at −140 mV is around 50% of I_K1(i.e. I_K1/I_f=0.5;FIG. 18C). Even for a cell with small I_f, automaticity could still be induced by I_K1suppression which reveals the latent pacing ability as demonstrated presently for the case of HCN-ΔΔΔ-transduced ACMs with hyperpolarized RMP in the presence of Ba²⁺ and previously with genetic suppression of I_K1(36). At the other extreme, excess I_foverwhelming I_K1(i.e. I_K1/I_f>0.5) would depolarize RMP to such an extend that automaticity cannot be induced.

Spontaneous generation of APs of ACMs occurred only when an optimal balance between I_fand I_K1was established. Therefore, a perturbation to this balance by changing either current would terminate pacing. This notion was supported by the experimental observations that 1) the firing activity of pacing Ad-CGI-HCN1ΔΔΔ-transduced ACMs (with I_K1/I_f=0.5) could be ceased after I_K1blockade (which was previously shown to unleash the latent pacemaker activity of ventricular CMs (15)), and 2) silent Ad-CGI-HCN1ΔΔΔ-transduced cells with hyperpolarized (I_K1/I_f<0.5) and depolarized (I_K1/I_f>0.5 RMP could be rendered electrically active by inhibiting I_K1and I_f(to increase and decrease I_K1/I_f), respectively. Thus, I_K1/I_fis crucial for inducing automaticity of ACMs by modulating the RMP, which subsequently affect voltage-gated Na⁺ and L-type Ca²⁺ currents that are involved in AP depolarization.

The present results have two major implications for future efforts on the generation of bio-artificial pacemaker. The first concerns the successful conversion of quiescent V or ACMs into pacemaker-like cells. Since an optimal operating ratio of I_fto I_K1exists, the dosage of I_fto be administered should be dependent upon the intrinsic I_K1expression that varies among chamber-specific cells. For instance, right ACMs (for SA node reconstruction) that have an intrinsically smaller I_K1density than that of VCMs would require less I_fexpression for conversion into pacing cells. Secondly, this work suggests the necessity for safety consideration in converted pacing cells for clinical application. I_fand I_K1mismatch not only fails to induce automaticity, but will result in heterogeneous RMP and APD in ACMs which may become a substrate for arrhythmias, thus further highlighting the importance of accurate and customized I_fdosing.

Collectively, it has been shown that a fine balance between I_fand I_K1is the key for successful induction of automaticity, and more importantly, its modulation in ACMs. A qualitative approach of I_foverexpression or I_K1suppression may be replaced with a more quantitative approach in titrating different ionic components, designed specifically for the target cells of interest to improve the current technology of bio-artificial pacemaker.

It is to be understood that while the invention has been described in conjunction with the above embodiments, that the foregoing description and examples are intended to illustrate and not limit the scope of the invention. Other aspects, advantages and modifications within the scope of the invention will be apparent to those skilled in the art to which the invention pertains.

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